We propose to continue with a detailed genetic analysis of Sindbis virus, a group A arbovirus (now called the alphavirus group of the togaviruses). These viruses consist of an icosahedral nucleocapsid containing the viral nucleic acid (a single-stranded RNA of molecular weight 4.3 million) surrounded by a lipoprotein envelope or membrane. The viral genome probably contains 10 to 12 cistrons, of which 8 are currently known. Of these 8 complementation groups, the function of 3 groups has been determined. Using temperature sensitive mutants already isolated we hope to enumerate the total number of cistrons by complementation tests and to determine the function of each gene. A wide variety of biochemical and biophysical assays will be used to study gene functions, including examination of RNA species made during infection, detailed analysis of mutant proteins produced, chemical and physical studies of mutant virions and nucleocapsids, temperature shift experiments to determine when during infection each function is necessary, and studies of the insertion of virus glycoproteins into the cell surface. We are studying in detail the replication and properties of one particular mutant which is defective in maturation and produces virus particles containing many nucleocapsids, and hope to identify and study other mutants defective in cell surface modification or virus assembly. We plan to compare the replication of Sindbis virus in established lines of mosquito cells with the life cycle in a mammalian or avian system, using ts mutants to delineate the functions necessary in the arthropod host.